The present invention relates to methods and pharmaceutical compounds and compositions for stimulating neurite outgrowth in nerve cells leading to nerve regeneration. More particularly, the compositions comprise compounds that inhibit the peptidyl-prolyl isomerase (rotamase) enzyme activity associated with the FK-506 binding protein (FKBP). The methods comprise treating nerve cells with compositions comprising the rotamase-inhibiting compound. The methods of the invention can be used to promote repair of neuronal damage caused by disease or physical trauma.
Immunophilins are a family of soluble proteins that serve as receptors for important immunosuppressant drugs such as cyclosporin A, FK-506 and rapamycin. An immunophilin of particular interest is the FK-506 binding protein (FKBP). For a review of the role of immunophilins in the nervous system, see Solomon et al., xe2x80x9cImmunophilins and the Nervous System,xe2x80x9d Nature Med., 1(1), 32-37 (1995).
The 12-kiloDalton FK-506 binding protein, FKBP12, binds FK-506 with high affinity. Such binding has been directly measured using microcalorimetry and radiolabeled FK-506, e.g., [3H]dihydro-FK-506 (see Siekierka et al., Nature, 341, 755-57 (1989); and U.S. Pat. No. 5,696,135 to Steiner et al.) and 32-[1-14C]-benzoyl-FK-506 (see Harding et al., Nature, 341, 758-60 (1989)). Binding affinity of other compounds for FKBP can be determined directly by microcalorimetry or from competitive binding assays using either tritiated or 14C-labelled FK-506, as described by Siekierka et al. or Harding et al.
FK-506-binding protein FKBP12 participates in a variety of significant cellular functions. FKBP12 catalyzes cis-trans isomerization of peptidyl-prolyl linkages. This peptidyl-prolyl isomerase enzyme activity is also referred to as rotamase activity. Such activity is readily assayed by methods known in the art (see Fischer et al., Biochim. Biophys. Acta 791, 87 (1984); Fischer et al., Biomed. Biochim. Acta 43, 1101 (1984); and Fischer et al., Nature 337, 476-478 (1989)). U.S. Pat. Nos. 5,192,773 and 5,330,993 to Armistead et al. report FKBP binding affinities that were correlated with rotamase-inhibiting activities for many compounds.
FK-506 and compounds that bind FKBP competitively with FKBP stimulate outgrowth of neurites (axons) in nerve cells (see U.S. Pat. No. 5,696,135 to Steiner et al.). Lyons et al. (Proc. Natl. Acad, Sci, USA, 91, 3191-95 (1994)) demonstrated that FK-506 acts to enhance or potentiate the effectiveness of nerve growth factor (NGF) in stimulating neurite outgrowth in a rat pheochromocytoma cell line. The mechanism of stimulation of such neurite outgrowth appears to be 10- to 100-fold potentiation of the action of nerve growth factor.
Potency for inhibition of the peptidyl-prolyl isomerase (rotamase) enzyme activity of FKBP by FK-506, and by compounds that competitively inhibit FK-506 binding to FKBP, empirically correlates with activity for stimulation of neurite outgrowth. Because of the close correlation between rotamase inhibition and neurotrophic action, it has been proposed that the rotamase may convert a protein substrate into a form that promotes neural growth (see U.S. Pat. No. 5,696,135). For example, it has been found that FKBP12 forms bound complexes with the intracellular calcium ion channelsxe2x80x94the ryanodine receptor (RyR) and the inositol 1,4,5-triphosphate receptor (IP3R) (Jayaraman et al., J. Biol. Chem., 267, 9474-9477 (1992); Cameron et al., Proc. Natl. Acad. Sci, USA, 92, 1784-1788 (1995)), helping to stabilize calcium release. For both the RyR and the IP3R, it has been demonstrated that FK-506 and rapamycin are capable of dissociating FKBP12 from these receptors. In both cases, the xe2x80x9cstrippingxe2x80x9d off of FKBP12 leads to increased leakiness of the calcium channels and lower intracellular calcium concentrations. It has been suggested that calcium flux may be associated with stimulation of neurite outgrowth.
In addition, FK-506-FKBP bound complexes bind to and inhibit calcineurin, a cytoplasmic phosphatase. The phosphatase activity of calcineurin is necessary for dephosphorylation and subsequent translocation into the nucleus of nuclear factor of activated T-cells (NF-AT) (see Flanagan et al., Nature, 352, 803-807 (1991)). NF-AT is a transcription factor that initiates interleukin-2 gene activation, which in turn mediates T-cell proliferation; these steps are important to the activation of an immune response. Calcineurin-inhibiting activity is correlated with the immunosuppressant activity of FK-506 and related compounds.
Calcineurin inhibition, however, does not correlate with the stimulation of neurite outgrowth. Therefore, compounds that are potent inhibitors of rotamase but not strong inhibitors of calcineurin are desired since they should be neurotrophic but non-immunosuppressive.
Such neurotrophic agents desirably find use in augmenting neurite outgrowth, and hence in promoting neuronal growth and regeneration in various pathological situations where neuronal repair can be facilitated, including peripheral nerve damage caused by injury or diseases such as diabetes, brain damage associated with stroke, and for the treatment of neurological disorders related to neurodegeneration, including Parkinson""s disease, Alzheimer""s disease, and amyotrophic lateral sclerosis (ALS). Further, such use is preferably without the associated effect of immunosuppression, since long-term use of immunosuppressants is associated with side effects such as kidney toxicity, neurological deficits, and vascular hypertension.
Various inhibitors of rotamase enzyme activity, FKBP-binding compounds, or immunomodulating compounds are known. See, e.g., U.S. Pat. Nos. 5,192,773, 5,330,993, 5,516,797, 5,612,350, 5,614,547, 5,622,970, 5,654,332, 5,665,774, 5,696,135, and 5,721,256. See also International Publication Nos. WO 96/41609, WO 96/40633, and WO 96/40140.
In view of the variety of disorders that may be treated by stimulating neurite outgrowth and the relatively few potent FKBP12-binding compounds that are known to possess this property, there remains a need for additional neurotrophic, rotamase-binding compounds. Such compounds will desirably have physical and chemical properties suitable for use in pharmaceutical preparations, e.g., bioavailability, half-life, and efficient delivery to the active site. In view of the desired properties, small organic molecules are preferred over proteins. Furthermore, such compounds will desirably lack significant immunosuppressive activity.
It is therefore an object of the invention to provide small-molecule neurotrophic agents. An additional object is to achieve rotamase-binding compounds that are non-immunosuppressive agents. It is a further object of the invention to provide effective processes for synthesizing such compounds as well as useful intermediates therefor. Another object of the invention is to provide methods for treating patients having neurological trauma or disorders as a result of, or associated with, conditions that include (but are not limited to) neuralgias, muscular dystrophy, Bell""s palsy, myasthenia gravis, Parkinson""s disease, Alzheimer""s disease, multiple sclerosis, ALS, stroke and ischemia associated with stroke, neural parapathy, other neural degenerative diseases, motor neuron diseases, and nerve injuries including spinal cord injuries.
Such objects have been achieved by the rotamase-binding agents of the present invention, which may be used to stimulate the growth and regeneration of neurons. The administration of these agents to individuals requiring therapeutic stimulation of neuronal growth and regeneration provides effective therapies in various pathological situations where neuronal repair can be facilitated, including peripheral nerve damage caused by injury or disease such as diabetes, brain damage associated with stroke, and for the treatment of neurological disorders related to neurodegeneration, including Parkinson""s disease, Alzheimer""s disease, and amyotrophic lateral sclerosis.
In one general embodiment, the rotamase-binding agents of the invention include compounds of the general structural formula (I-a): 
wherein:
R1 is selected from hydrogen, substituted and unsubstituted alkyl, alkenyl, aryl, C3-C8 cycloalkyl, and C5-C7 cycloalkenyl groups, and C(R11)(R12)(R13), the alkyl and alkenyl groups being optionally substituted with C1-C4 alkyl, C2-C4 alkenyl, C4-C6 cycloalkenyl, or hydroxy, the aryl group being optionally substituted with halogen, hydroxyl, NO2, CF3, C1-C6 alkyl, C2-C6 alkenyl, C1-C4 alkyloxy, C2-C4 alkenyloxy, benzyloxy, phenoxy, amino, or phenyl, and the cycloalkyl and cycloalkenyl groups being optionally substituted with C1-C4 alkyl, C2-C4 alkenyl, C1-C4 alkyloxy, or hydroxy, and R11 and R12 each independently being lower alkyl, or R11 and R12 together with the atom to which they are bound forming cycloalkyl, and R13 being H, OH, lower alkyl, aryl, or (CH2)nxe2x80x94Oxe2x80x94W1, where n is 0, 1, 2, or 3, W1 is R2 or C(O)R2, and R2 is C1-C3 alkyl optionally substituted with one or two methoxy groups;
X is selected from hydrogen, cyano, C1-C2 alkyloxy, dimethoxymethyl, and oxygen, where when X is oxygen, the Cxe2x80x94X bond (i.e., the bond connecting X to the ring carbon atom) is a double bond; and
Y is selected from hydrogen, alkyl, alkenyl, and cycloalkyl, the alkyl, alkenyl, and cycloalkyl groups being optionally substituted in one or more positions with substituents selected from substituted and unsubstituted alkyl, aryl, alkoxy, hydroxyalkyl, aryloxy, alkenyloxy, hydroxy, (CH2)pxe2x80x94Oxe2x80x94W2, and (CH2)pxe2x80x94Nxe2x80x94W2, where p is 0, 1, or 2, and W2 is R3 or C(O)R3, where R3 is alkyl, alkenyl, or aryl optionally substituted with alkyl, aryl, or alkoxy; or
X and Y taken together with the nitrogen heteroatom of the ring structure to which Y is connected (shown in formula (I-a)) form a 5- to 7-membered saturated or unsaturated heterocyclic ring optionally containing one additional heteroatom (i.e., one heteroatom in addition to the depicted nitrogen atom of the ring structure) selected from 0 and N, the 5- to 7-membered saturated or unsaturated heterocyclic ring being optionally substituted with one or more substituents selected from J, K, and L, which are independently oxygen, C3-C5 cycloalkyl, or C1-C5 alkyl optionally substituted with one or two substituents independently selected from C3-C5 cycloalkyl, methoxy, methoxyphenyl, or dimethoxyphenyl, or J and K taken together form a phenyl ring optionally substituted with one or more substituents selected from methoxy, trifluoromethyl, trifluoromethoxy, and suitable substituents linked to the phenyl ring through oxygen, nitrogen, carbon, or sulfur.
In an alternative general embodiment, the invention is directed to compounds of formula (I-b): 
wherein:
R1 is selected from hydrogen, substituted and unsubstituted alkyl, alkenyl, aryl, C3-C8 cycloalkyl, and C5-C7 cycloalkenyl groups, and C(R11)(R12)(R13), the alkyl and alkenyl groups being optionally substituted with C1-C4 alkyl, C2-C4 alkenyl, C4-C6 cycloalkenyl, or hydroxy, the aryl group being optionally substituted with halogen, hydroxyl, NO2, CF3, C1-C6 alkyl, C2-C6 alkenyl, C1-C4 alkyloxy, C2-C4 alkenyloxy, benzyloxy, phenoxy, amino, or phenyl, and the cycloalkyl and cycloalkenyl groups being optionally substituted with C1-C4 alkyl, C2-C4 alkenyl, C1-C4 alkyloxy, or hydroxy, and R11 and R12 each independently being lower alkyl, or R11 and R12 together with the atom to which they are bound forming cycloalkyl, and R13 being H, OH, lower alkyl, aryl, or (CH2)nxe2x80x94Oxe2x80x94W1, where n is 0, 1, 2, or 3, W1 is R2 or C(O)R2, and R2 is C1-C3 alkyl optionally substituted with one or two methoxy groups;
X1 and X2 are each independently selected from hydrogen, cyano, C1-C2 alkyloxy, dimethoxymethyl, and oxygen, where when X1 or X2 is oxygen, the Cxe2x80x94X bond (i.e., the bond connecting X1 or X2 to the ring carbon atom) is a double bond (i.e, X1 or X2 is xe2x95x90O); or X1 and X2 together form a valence bond; and
Y is selected from hydrogen, alkyl, alkenyl, and cycloalkyl, the alkyl, alkenyl, and cycloalkyl groups being optionally substituted in one or more positions with substituents selected from substituted and unsubstituted alkyl, aryl, alkoxy, hydroxyalkyl, aryloxy, alkenyloxy, hydroxy, (CH2)pxe2x80x94Oxe2x80x94W2, and (CH2)pxe2x80x94Nxe2x80x94W2, where p is 0, 1, or 2, and W2 is R3 or C(O)R3, where R3 is alkyl, alkenyl, or aryl optionally substituted with alkyl, aryl, or alkoxy; or
one of X1 and X2 in combination with Y taken together with the nitrogen heteroatom of the ring structure to which Y is connected (shown in formula (I-b)) form a 5- to 7-membered saturated or unsaturated heterocyclic ring optionally containing one additional heteroatom (i.e., one heteroatom in addition to the depicted nitrogen atom of the ring structure) selected from O and N, the 5- to 7-membered saturated or unsaturated heterocyclic ring being optionally substituted with one or more substituents selected from J, K, and L, which are independently oxygen, C3-C5 cycloalkyl, or C1-C5 alkyl optionally substituted with one or two substituents independently selected from C3-C5 cycloalkyl, methoxy, methoxyphenyl, or dimethoxyphenyl, or J and K taken together form a phenyl ring optionally substituted with one or more substituents selected from methoxy, trifluoromethyl, trifluoromethoxy, and suitable substituents linked to the phenyl ring through oxygen, nitrogen, carbon, or sulfur.
The rotamase-inhibiting agents of the invention also include pharmaceutically acceptable derivatives of such compounds of the formula (I-a) or (I-b).
The invention further relates to methods of treating neurological trauma or disorders as a result of, or associated with, conditions including neuralgias, muscular dystrophy, Bell""s palsy, myasthenia gravis, Parkinson""s disease, Alzheimer""s disease, multiple sclerosis, amyotrophic lateral sclerosis (ALS), stroke and ischemia associated with stroke, neural parapathy, other neural degenerative diseases, motor neuron diseases, and nerve injuries including spinal cord injuries. The inventive methods comprise administering a therapeutically effective amount of a compound of formula (I-a) or (I-b), or a prodrug, pharmaceutically active metabolite, or pharmaceutically acceptable (nontoxic) salt thereof, to a patient in need of such treatment. Such methods further comprise administering a composition comprising an effective amount of a compound of formula (I-a) or (I-b), or a prodrug, pharmaceutically active metabolite, or pharmaceutically acceptable salt thereof, in combination with a pharmaceutically acceptable carrier or diluent and/or a therapeutically effective amount of a neurotrophic factor selected from nerve growth factor, insulin growth factor and its active truncated derivatives, acidic and basic fibroblast growth factor, platelet-derived growth factors, brain-derived neurotrophic factor, ciliary neurotrophic factors, glial cell line-derived neurotrophic factor, neurotrophin-3, and neurotrophin 4/5, to a patient in need of such treatment.
The invention also relates to intermediates of formulae (II), (III), and (V), which are described below and are useful for preparing the FKBP-modulating compounds of formula (I-a) and (I-b). The invention further relates to processes of making the compounds using such intermediates.